Abstract: OBJECTIVE To investigate the relationship between the respiratory tract sputum flora and the clinical TNM staging of non-small cell lung cancer (NSCLC) in patients. METHODS Sixty-five NSCLC patients treated at the Second Affiliated Hospital of Nantong University from Jan. 2020 to Oct. 2022 and twenty-five healthy controls (HC) during the same period were selected as study subjects. Among them, the 65 NSCLC patients were divided into an early stage (ES) group with 30 cases (stages I-II) and an advanced stage (AS) group with 35 cases (stages III-IV) based on TNM staging. Sputum samples from study subjects were collected and analyzed for bacterial composition based on 16S rRNA gene sequencing. RESULTS In α diversity analysis, compared with the HC group, the Chao 1 index increased in both ES and AS groups (t=5.397, 2.838, P＜0.001, 0.017), and the Shannon index decreased in the AS group compared with the ES group (t=4.337, P=0.008). In β diversity analysis, differences in microbiota were observed among the three sputum samples (P＜0.05). The AS group had a higher relative abundance of Firmicutes and a lower relative abundance of Bacteroidetesthan the HC and ES groups (P＜0.05) . Compared with the HC and ES groups, the AS group showed a decrease in the relative abundance of Prevotella and Prevotella (type 7) (P＜0.05), and an increase in the relative abundance of Streptococcus and Rothia (P＜0.05). At the genus level, the ES group was enriched with genera including Atopobium and Actinomyces, while the AS group was enriched with genera including Romboutsia and Rothia. Compared with the ES group, the AS group exhibited significant upregulation of microbial genes related to cell signaling, xenobiotic biodegradation and metabolism and amino acid metabolism, while significant downregulation of microbial genes related to genetic information processing, translation and metabolism. CONCLUSIONS There are significant differences in the taxonomic structure of sputum microbiota between early and late-stage NSCLC patients, and specific airway microbial level changes are possibly involved in the pathophysiological processes of NSCLC development.